EXCEED THE SPACE PROVIDED. One of the most exciting advances in understanding the molecular actions of growth hormone (GH) was the identification of the GH receptor (GHR) associated tyrosine (Tyr) kinase JAK2 as an essential signaling molecule for GH. Using techniques and reagents developed and phosphorylated Tyr (pTyr) and JAK2 binding proteins identified during this MERIT award, additional novel insight into how GH elicits its diverse responses will be obtained by continuing to test the hypothesis that activation of JAK2 in response to GH results in the phosphorvlation of multiple Tyr in JAK2 and GHR that serve as regulatory sites or binding sites for molecules in various GH signaling pathways. Aim 1will continue to systematically map the JAK2 phosphorylation sites in JAK2 and GHR that are regulated by GH using 2-D peptide mapping, mass spectrometry, and phosphospecific antibodies (Ab). Aim 2 will examine whether phosphorylation of specific Tyr in JAK2 regulates JAK2 activity. Aim 3 will determine whether specific pTyr in JAK2 and/or GHR are required for activation of specific genes using JAK2- and GHR-deficient cell lines that stably express GHR and JAK2 with single Y-[unreadable]Fmutations, Affymetrix microarrays, real time quantitative polymerase chain reaction and/or promoter reporter constructs. Aim 4 will identify pTyr in JAK2 and/or GHR required for activation of specific GH signaling pathways. Aim 5 will continue to identify and characterize new signaling molecules that interact with pTyr within JAK2 and/or GHR. Previously identified JAK2 interacting proteins that appear to be potent inhibitors of GH signaling, SSG1 and TM6P1, will be further characterized. Aim 6 will characterize new JAK2 binding domains, including the newly identified SRPX5 (02)JAK2 binding domain in SSG1. This unbiased, global, and rigorous approach will provide needed insight into: 1) the initiating steps in GHR signal transduction; 2) new signaling proteins and pathways utilized by GH; 3) novel genes regulated by GH; 4) the mechanisms by which these pathways and genes are initiated/regulated; 5) pathways that interact, either because they compete for the same binding site in GHR and/or JAK2, lie downstream of a common initial signaling molecule or require input from multiple upstream pathways; and 6) novel JAK2, potential pTyr, binding domains. This insight will facilitate delineating the molecular mechanisms by which GH elicits its diverse effects on growth and metabolism and ultimately guide the design of therapeutic agents that target specific GH-regulated growth and metabolic pathways. Because JAK2 is a key signaling protein used by many other ligands that bind to cytokine family receptors, including leptin, prolactin, many interleukins and interferons, the findings of this study should provide valuable insight into many human physiological functions and their associated diseases, including metabolic syndrome, obesity and a variety of diabetic complications.